In-package pasteurization of ready-to-eat (RTE) meals using microwave-assisted pasteurization system (MAPS) has shown promise for improving the safety and quality of foods, since dielectric heating is more efficient than thermal conduction. However, MAPS can affect the oxygen transmission rate (OTR) and water vapor transmission rates (WVTR) of packaging, imparting a certain degree of quality deterioration to the pasteurized food. This study evaluates morphological changes in newly-developed 3 multilayer polymeric films used for lid-stock on trays subjected to MAPS. We measured changes in OTR and WVTR after MAPS treatment, and further correlated these measurements with melting enthalpy (ΔH), overall crystallinity, crystal structure, water absorption, and dielectric properties of films. The composition of tested films was: (Film A): polyethylene terephthalate (PET)/barrier PET/polyethylene (PE); (Film B): PET/Nylon-Polypropylene (PP) and (Film C): PET/low density polyethylene (LDPE)/Nylon/LDPE. Results show that the OTR and WVTR of the films significantly increased (P < 0.05) after both hot water and MAPS pasteurization. Films B and C exhibited a higher OTR after MAPS (52 mins) compared to hot water pasteurization (36 mins). The melting enthalpy (ΔH) of the films increased after pasteurization, and was correlated to the increase in overall crystallinity (1–4% increase) of the films. Increases in OTR and WVTR can be attributed to the fragmented crystal structures and smaller crystal size observed in X-ray diffraction. It is evident that the films absorbed water during MAPS, which altered their dielectric properties. In addition, it is likely that water absorption caused plasticization of the Nylon polymer, degrading the gas barrier properties of the film. Based on these findings, we recommend using a multilayer film with PET as barrier layer for MAPS treatment. Commercialization of novel microwave-assisted thermal pasteurization technology will require development of multilayer films which can sustain microwave and thermal stresses. Food processes influence oxygen and water vapor barrier properties of films which may affect the shelf-life of food sensitive to oxygen and water vapor. A better understanding of the influence of microwave-assisted thermal pasteurization process on film properties will help polymer industry in the design and development of improved film structures.